Majority of the proteome is constituted by oligomers and their function is governed by underlying protein-protein interactions. Interfacial residues, namely residues right at the interface of two protein chains, are known to confer stability and specificity in dimers. However, other interactions play a significant role in the formation and maintenance of oligomers in protein assemblies as well. Inter-protein bifurcated interactions are those where one residue simultaneously interacts with two residues belonging to two neighbouring protein chains. The characteristic features for such higher order interactions remain largely unexplored and unknown. In this study, we focused on r esidues specifically involved in b ifurcated i nteractions (referred as RBI). We examine the bifurcated inter-protein interactions by assembling a dataset of protein assemblies of known 3D structures. We have characterized the type of interactions and the residues involved in the interactions using parameters like energy contributions and conservation score. We find that the residues participating in bifurcated inter-protein interactions contribute more to the stability of the complex than other interfacial residues. Furthermore, we have presented examples where mutation of a residue involved in a bifurcated interaction results in detrimental outcomes. This study highlights the significance of inter-protein bifurcated interactions that contribute to the stability of multiple interfaces in protein oligomers and hence contribute to the expansion of the understanding of protein assemblies.

Toll-like receptors (TLRs) are major players of the innate immune system – recognizing pathogens and differentiating self/non-self components of immunity. These proteins are present either on the plasma membrane or endosome and recognise pathogens at their extracellular domains. They are also characterised by a single transmembrane helix and an intracellular TIR domain. Few TIRs directly invoke downstream signalling, while others require other TIR domains of adaptors like TRAM and TRIF. On recognizing pathogenic lipopolysaccharides (LPS), TLR4 dimerises and interacts with the intracellular TRAM dimer through the TIR domain to further recruit TRIF molecules. We have performed an in-depth study of the effect of two mutations, P116H and C117H, at the dimeric region of the adaptor TRAM, which are known to abrogate downstream signalling. We modelled the structure and performed molecular dynamics studies to infer the structural changes occurring across the trajectory due to the point mutations in order to decipher the structural basis of this dramatic effect. We observed that these mutations led to increased RoG (Radius of Gyration) of the complex and resulted in several changes to the interaction energy values when compared against the wild type and few positive control mutants. We identified highly interacting residues as hubs and few such hubs that were lost in the mutant dimers. Further, changes in the protein residue path, hampering the information flow between the crucial AEDD and TS sites, happen for the mutants. Overall, we show that such residue changes can have subtle but long-distance effects, impacting the signaling path allosterically.

Proteins such as enzymes perform their function by predominant non-covalent bond interactions between transiently interacting units. There is an impact on the overall structural topology of the protein, albeit transient nature of such interactions, that enable proteins to deactivate or activate. This aspect of the alteration of the structural topology is studied by employing protein structural networks, which are node-edge representative models of protein structure, reported as a robust tool for capturing interactions between residues. Several methods have been optimised to collect meaningful, functionally relevant information by studying alteration of structural networks. In this article, different methods of comparing protein structural networks are employed, along with spectral decomposition of graphs to study the subtle impact of protein-protein interactions. A detailed analysis of the structural network of interacting partners is performed across a dataset of around 900 pairs of bound complexes and corresponding unbound protein structures. The variation in network parameters at, around and far away from the interface are analysed. Finally, we present interesting case studies, where an allosteric mechanism of structural impact is understood from communication-path detection methods. The results of this analysis are beneficial in understanding protein stability, for future engineering and docking studies.

Protein domains are structural, functional, and evolutionary units. These domains bring out the diversity of functionality by means of interactions with other co-existing domains and provide stability. Hence, it is important to study intra-protein inter-domain interactions from the perspective of types of interactions. Domains within a chain could interact over short timeframes or permanently, rather like protein-protein interactions (PPIs). However, no systematic study has been carried out between two classes, namely permanent and transient domain-domain interactions (DDIs). In this work, we studied 264 two-domain proteins, belonging to either of these classes and their interfaces on the basis of several factors, such as interface area and details of interactions (number, strengths, and types of interactions). We also characterized them based on residue conservation at the interface, correlation of residue motions across domains, its involvement in repeat formation, and their involvement in particular molecular processes. Finally, we could analyse the interactions arising from domains in two-domain monomeric proteins, and we observed significant differences between these two classes of domain interactions and a few similarities. This study will help to obtain a better understanding of structure-function and folding principles of multi-domain proteins.